Polycyclic aromatic hydrocarbons (PAHs) such as benzo[a]pyrene (BaP) and benzo[c]phenanthrene (BcPh) are widespread environmental pollutants, many of which are potent carcinogens in mammals. These hydrocarbons are metabolically activated through the action of cytochrome P450 and epoxide hydrolase to give highly reactive bay-region diol epoxides (DEs) which form covalent DNA adducts, mainly at the exocyclic amino groups of purine bases. Erroneous replication of the modified residues by DNA polymerases leads to mutations, which represent a likely first step in the induction of cancer. Sixteen bay-region DE-purine adducts are metabolically possible for a given parent hydrocarbon. Duplex oligonucleotides containing these adducts exhibit distinct structural motifs and thus provide unique tools to probe the catalytic and recognition sites of DNA-processing enzymes. Previous methods for preparing site-specifically adducted oligonucleotides were limited by low yields (in the 50 microgram range) as well as the inability to place adducts in biologically relevant sequences containing multiple purine nucleotide residues. We have developed powerful new synthetic methods which make possible the relatively facile synthesis of adducted nucleosides and site-specifically adducted oligonucleotides in any desired sequence contest on 3-5 milligram scale, suitable for NMR and X-ray crystallographic studies as well as for biochemical investigations. Current synthetic efforts focus on stereoselective synthesis of phosphoramidite building blocks derived from dA and dG adducts of dibenzo[a,l]pyrene diol epoxides (DBalP DEs), the most carcinogenic PAH DEs identified to date, and their incorporation into oligonucleotides. In the course of preparing a duplex oligonucleotide containing a trans opened DBalP DE adduct at the central G of a 5?-CCATCGTACC-3? sequence, we made the surprising observation (1) that the hydrocarbon moiety migrated from the strand to which it was covalently bound to the complementary strand to give two new adducted products: a major product with the hydrocarbon on the G complementary to the C immediately 5? to the original adduct, and a minor product with hydrocarbon on G complementary to the C immediately 3? to the original adduct. Approximately half of the hydrocarbon had migrated to the complementary strand after six days at room temperature and neutral pH. Inversion of configuration at the point of attachment of the hydrocarbon to the DNA was observed in the migration products. The ability of a PAH adduct to ?jump? from one strand to the other in duplex DNA is unprecedented and suggests a completely new pathway for mutagenesis. We anticipate that NMR studies currently in progress on the adducted oligonucleotide duplex will reveal structural features responsible for this unique reaction.[unreadable] [unreadable] Enzymology of PAH DE Adduct Processing: Enzymes currently under study include human DNA polymerase beta, vaccinia viral topoisomerase I, Werner syndrome helicase and HIV-1 integrase. i) DNA polymerase beta (Pol beta) functions as a nucleotidyl transferase to fill gaps in one strand of double-stranded DNA that result from base excision of DNA damage followed by endonuclease cleavage at the abasic site. We have determined the crystal structure of human Pol beta bound to a 1-nucleotide gapped DNA containing a BcPh DE-deoxyguanosine (dG) adduct in the templating position opposite the gap (2). In this structure, the hydrocarbon portion of the adduct stacks over the base pair immediately 5? to the modified guanine and blocks the normal binding site for the incoming deoxynucleoside triphosphate. Thus, we observe that correct insertion of dC opposite the adduct is more than 6 orders of magnitude less efficient than opposite a normal dG template. In our structure, the modified dG assumes a syn conformation which can hydrogen bond to an incoming dATP or dGTP. Consistent with this structure, misinsertion of purine nucleotides opposite the adducted dG is preferred over insertion of the correct dC. ii) Topoisomerase I of vaccinia, a poxvirus closely related to the virus that causes smallpox, is an enzyme that relaxes supercoiled DNA. The enzyme makes a transient single-strand break in duplex DNA to give a phosphotyrosyl bond to a DNA 3'-phosphate group and a free 5?-hydroxyl group on the other side of the break. Chemically intact but relaxed DNA is formed and the free tyrosyl enzyme is regenerated in a second, religation step. In previous studies we systematically mapped topoisomerase-substrate contacts at the vaccinia enzyme?s recognition and cleavage sites by observing the effects of PAH-DE adducts of defined stereochemistry on the rate and extent of the cleavage step. BaP DE-dA adducts that intercalate into the DNA at or near the cleavage site permit cleavage, although they significantly retard its rate. We have now determined that such intercalating adducts inhibit the religation step by increasing the amount of enzyme-substrate covalent complex at equilibrium by a factor of ~10- to 15-fold, thereby trapping and ?poisoning? the topoisomerase (3). Understanding of the structural requirements for irreversible poisoning of this essential viral enzyme could lead to design of novel antiviral agents. iii) Helicases are required for unwinding of duplex DNA, and may have a role in repairing stalled replication forks in the presence of damaged DNA. Deficiencies in these enzymes result in genomic instability disorders such as Werner and Bloom syndromes. Werner syndrome helicase unwinds DNA by translocating along one strand in the 3?- to 5?-direction and displacing the opposite strand. We have found (4) that BaP dG adducts located on the strand on which the helicase translocates inhibit DNA unwinding in a manner that does not depend of their stereochemical orientation. In contrast, these dG adducts have no effect on DNA unwinding when located on the displaced strand. iv) We are continuing to explore the effects of PAH adducts and other DNA modifications on DNA cleavage and strand transfer reactions catalyzed by HIV-1 integrase. Integrase first cleaves a dinucleotide (GT) fragment from the 3?-end of one DNA strand (leaving a 2-nucleotide overhang on the opposite strand), and then catalyzes insertion of the cleaved end into another strand of DNA. We have observed (5) that intercalating BaP DE-dA adducts at or near the 3?-cleavage site of an oligonucleotide substrate have no effect on the cleavage reaction, whereas minor-groove bound BaP DE-dG adducts 1 or 2 bases 5? to the cleavage site inhibit 3?-cleavage to an extent that depends on their stereochemical orientation relative to the cleavage site. A BaP adduct on the cleaved GT dinucleotide was found to inhibit strand transfer, consistent with the interpretation that this fragment remains associated with the enzyme during the strand transfer step (5).